Acute coronary syndrome (ACS) is a leading cause of death in the world. ACS is an umbrella term used to cover any group of clinical symptoms compatible with acute myocardial ischemia. Acute myocardial ischemia is chest pain due to insufficient blood supply to the heart muscle that results from coronary artery disease (also called coronary heart disease).
Platelets are known to play an important role in ACS. Platelets, or thrombocytes, are small, irregularly-shaped anuclear cells (i.e., cells that do not have a nucleus containing DNA), 2-4 μm in diameter, which are derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is between 8 and 12 days. Platelets play a fundamental role in hemostasis and are a natural source of growth factors. They circulate in the blood of mammals and are involved in hemostasis, leading to the formation of blood clots.
If the number of platelets is too low, excessive bleeding can occur (hemorrhage). However, if the number of platelets is too high, blood clots can form (thrombosis), which may obstruct blood vessels and result in such events as a stroke, heart attack, pulmonary embolism or the blockage of blood vessels to other parts of the body, such as the extremities of the arms or legs.
The function of platelets is the maintenance of haemostasis. This is achieved primarily by the formation of platelet aggregate, when damage to the endothelium of blood vessels occurs. On the converse, thrombus (platelet aggregate in patho-physiological condition) formation must be inhibited at times when there is no damage to the endothelium. Thrombus formation is generally associated with activation of the platelets.
Platelet activation involves changes to platelet metabolic biochemistry, shape, surface receptors, and/or membrane phospholipid orientation. Platelet activation involves a series of morphologic and functional changes in the platelets. Changes to platelet metabolic biochemistry result from substances generated by the platelet itself as well as cells in the blood vessels. Substances that induce platelet activation are called agonists. Each agonist attaches to a specific receptor on/inside the platelet, causing a series of reactions inside of the platelet.
The inner surface of blood vessels is lined with a thin layer of endothelial cells. Under this is a layer of collagen. Under normal physiological conditions, collagen does not pass into the bloodstream as endothelial cells produce a protein called von Willebrand factor (vWF), a cell adhesion ligand, which helps endothelial cells adhere to collagen. When the endothelial layer is injured, the collagen is exposed.
When the platelets contact collagen, they are activated. They are also activated by thrombin and adenosine diphosphate (ADP). They can also be activated by a negatively-charged surface, such as glass.
Platelet activation further results in the scramblase-mediated transport of negatively-charged phospholipids to the platelet surface. These phospholipids provide a catalytic surface (with the charge provided by phosphatidylserine and phosphatidylethanolamine) for the tenase and prothrombinase complexes.
Activated platelets change in shape to become more spherical, and pseudopods form on their surface. Thus they assume a stellate (star-like) shape.
Following platelet activation, platelets can aggregate, or clump together to form a hemostatic plug, using fibrinogen and vWF as a connecting agent. Platelet adhesion is generally the initial step in the formation of the hemostatic plug. Activated platelets will adhere, via glycoprotein (GP) Ia, to the collagen that is exposed by endothelial damage. Aggregation and adhesion act together to form the platelet plug. The most abundant platelet aggregation receptor is glycoprotein (GP) IIb/IIIa; this is a calcium-dependent receptor for fibrinogen, fibronectin, vitronectin, thrombospondin, and von Willebrand factor (vWF). Myosin and actin filaments in platelets are stimulated to contract during aggregation, further reinforcing the plug.
Clinical trials have shown that aspirin and clopidogrel help prevent myocardial infarction, stroke, and cardiovascular death. Aspirin and clopidogrel are believed to inhibit the production of chemicals responsible for platelet activation. For example, an activator and mediator of many leukocyte functions, including platelet aggregation, inflammation, and anaphylaxis, is platelet-activating factor, a potent phospholipid also known as a PAF, PAF-acether or AGEPC (acetyl-glyceryl-ether-phosphorylcholine).
For normal subjects that do not exhibit ACS, platelets are inactivated and would show resting behavior (unless the subject is emotionally stressed or in any other abnormal physiological condition). On the other hand, for subjects who are ACS risk prone, but unaware of it and not taking aspirin and/or clopidogrel, the platelets will be in the activated state. The subjects who are ACS risk prone, but aware of it and taking aspirin and/or clopidogrel, the platelets would likely be in a lowered activated state if the subject is not resistant to aspirin and/or clopidogrel.
Clopidogrel is recommended for the management of ACS. This along with aspirin, is recommended in the American College of Cardiology/American Heart Association guideline. It is also used along with aspirin, during the placement of coronary artery stents. Clopidogrel resistance was recognized in such procedures, as several patients did not have the anticipated platelet aggregation response to an ex vivo adenosine diphosphate challenge. This assessment can be made by individual aggregometric studies. Failure to obtain a deaggregation profile in a patient treated with clopidogrel at a reasonable ADP concentration (10 um) would normally considered as a signature for drug resistance.
From the EXCELSIOR study, which investigated the phenomenon, it was appreciated that it was present prior to treatment with clopidogrel and was therefore an intrinsic property of the patient's platelet (information is available on the worldwide web).
A study relating to clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE) revealed a statistically significant 8.7% (P=0.043) relative risk reduction in stroke, myocardial infarction, or ischaemic death in patients treated with clopidogrel in comparison to aspirin. The resistance to either of the drugs is therefore an important field of study. The dosage specification of the drugs also varies in different countries as different doses are recommended. The variation in population makes the proper assessment of drug resistance variability in population difficult.
In many cases dual antiplatelet drug therapy is used for patient safety. Despite the benefits of dual antiplatelet therapy, many patients continue to suffer adverse consequences (thrombus formation) of cardiovascular disease as they may be actually resistant to such drugs. It is thus important to have a quick sensor that will firstly assess the resistance to aspirin or clopidogrel in patients in one step and secondly assess the equivalence of the drug effects with respect to variations in geographic populations (which may correspond to genetic variations of patient population) and variations in licensed manufacturer of the drugs.
The conventional kit of drug resistance (e.g., using the PLT VASP/P2Y12 Test kit manufactured by BD) as described in Aleil et al. J. Thromb Haemost. 2005, 3, 85-92, is specific to clopidogrel resistance.
Corgenix Medical (CONX), a Denver diagnostic test-kit maker, has developed AspirinWorks to help doctors identify those not likely to benefit from a daily aspirin dose. A report on the test's efficacy was presented in July by independent Canadian and Australian researchers at a meeting of the International Society on Thrombosis & Haemostasis.
Neither of these kits can, however, find out whether there is single or multiple drug resistance as they are specific to individual drug. Therefore, it would be desirable to develop a method that may enable a quick assessment of (a) a patient's drug resistance single or multiple drugs, (b) a subject's risk assessment to ACS and (c) equivalence of drug potency with respect to geographic genetic or manufacturer variations.